Typically, MSMS is not used in the art to routinely quantitate proteins. The most common method used for protein detection/quantitation is still immunoassay/ELISA, particularly in clinical settings. Triple quadrupole MSMS is most usually employed for the analysis of small molecules. This is because the molecular weight for effective analysis is limited to approximately 3,000 Daltons. For this reason, the technique is often deemed unsuitable for analysis of proteins which may have much larger masses. Although some other MS techniques may be suitable for proteins, such as MALDI-TOF, MSMS is required for specificity. In order to overcome the difficulties in analysing proteins whose molecular weight exceeds 3,000, these are split into peptides. Relatively short peptides, for example peptides of 8 amino acids, can provide a virtually specific identification of a protein.
In a further development, it is desired to use MSMS for quantitative measurements of protein. The prior art MSMS systems feature only qualitative analysis. At best, prior art attempts have only achieved a semi-quantitative result. In order to achieve quantitation, ideally the whole protein needs to be labelled. This has been attempted in connection with, Apolipoprotein E4. This protein, was produced in an expression system. This is a very expensive and labour intensive technique. The cost of this technique is such that it is prohibitive for routine use.
In MS (such as electrospray MSMS) there are problems related to interference, especially through ion suppression effects. Ion suppression results from the presence of less volatile compounds that can change the efficiency of droplet formation or droplet evaporation, which in turn affects the amount of charged ion in the gas phase that ultimately reaches the detector.
Thus, an important factor that can affect the quantitative performance of a mass detection is ion suppression. Sample matrix, coeluting compounds, and other factors can contribute to this effect. Ionization effects can theoretically occur in either the solution phase or the gas phase. The mass and charge of individual analytes are factors in making a compound a candidate for ion suppression or in making one compound a source of ion suppression for another. It has been shown that molecules with higher mass will suppress the signal of smaller molecules and that more polar analytes are more susceptible to suppression.
The presence of ion suppression or other deleterious effects can be evaluated via experimental protocols. The first involves comparison of (a) the instrument response for calibrators (including any internal standards) injected directly in mobile phase, (b) the same amount of compound added to preextracted samples, and (c) the same amount of compound added to specimen matrix before extraction. The second protocol, which can be viewed as part of interference checks for an assay, involves injection of drugs or metabolites that may also be present in the specimen. Just because a coeluting drug does not produce similar mass fragments does not mean that this compound is incapable of ion suppression.
Another problem with MSMS in the analysis of proteins is a problem of digestion. Typically, the peptides are generated from the protein by endopeptidase action. The industry standard endopeptidase for use in this application is trypsin. Clearly, an efficient or complete trypsin digestion is required in order to successfully reduce the protein to its component peptides for MSMS analysis. For quantitative analysis, it is important that an indication of the digestion is obtained in order to validate the readout of the individual peptide fragments from the target protein. In order to address this problem Anderson and Hunter (2006 Mol Cell Proteomics vol 5 pp 573-88) cloned DNA sequences for clinically important peptides, linked them, and expressed them in a cell free protein expression system with stable isotope arginine and lysine added. This results in a labelled fusion protein consisting of a series of concatenated peptides which should be theoretically separated by the action of trypsin. A problem with this approach is that the concatenated peptides are not equivalent in secondary or tertiary structure to any of the parent polypeptides. A further problem with this approach is that cleavage of the protein is very likely to be sterically hindered by the large number of peptides which have been fused together. Furthermore, the trypsin cleavage site for many of these peptides will be masked in the three dimensional structure of a large fusion protein. Even if these sites can eventually be cleaved, it is likely to be at a low efficiency and perhaps with reaction kinetics which might interfere with the analysis. In addition, Anderson et al report that in use only a minor proportion of the predicted tryptic digest products can be detected (17/30 such peptides were not reproducibly detected according to Anderson). Furthermore, this multi-fused peptide labelling is difficult to perform according to Anderson. Even if this could be reliably reproduced, it still involves a cell free system for the production of the fusion protein. Such systems can suffer from metabolism of lysine or arginine or other amino acids. This makes it more difficult to control the range of products which are produced. Moreover, there is a significant error rate causing introduction of incorrect amino acids into the fused polypeptide product. This is caused at the translation stage so that the mRNA which is introduced may not be accurately translated into protein in such a cell free system, leading to errors in the polypeptide which can be variable and can be as high as several percent of polypeptide product.
Even where this approach has been optimised, the tryptic peptide fragments are typically prepared in parallel, separate from the digestion of the sample to be analysed, and are then ‘spiked’ into the sample before analysis. Clearly, such an approach is incapable of controlling for endopeptidase action in the sample to be analysed.
The present invention seeks to overcome problems associated with the prior art.